超低温冷冻技术在滇池金线鲃种质资源保存中的应用
文献类型：学位论文
| 作者 | 王晓爱 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 杨君兴,陈小勇 |
| 关键词 | 滇池金线鲃 细胞培养 精子冷冻 精子质量评价 |
| 其他题名 | The application of cryopreservation on germplasm preservation of Sinocyclecheilus grahami |
| 学位专业 | 动物学 |
| 中文摘要 | 滇池金线鲃Sinocyclocheilus grahami隶属于鲤形目Cypriniformes鲤科Cyprinidae鲃亚科Barbinae，是滇池流域的特有种。因栖息地破坏，滇池金线鲃数量锐减，而被列为国家二级保护动物。为了保护这一珍稀物种，一直致力于研究其人工繁殖，并在2007年突破其人工繁殖技术，这是继中华鲟和胭脂鱼后，成功实现人工繁殖的第三种国家级保护鱼类。滇池金线鲃人工增殖放流的成功，在个体和群体水平上保护了这一珍稀物种，但细胞水平的研究还为开展。为此，我们研究了滇池金线鲃的细胞培养和精子冷冻保存技术，在细胞水平上保存其种质资源，并为其生产实践和大规模生产提供参考。 借鉴哺乳动物和其他鱼类的细胞培养方法，（1）建立了适合滇池金线鲃的细胞培养方案，并建立了7株滇池金线鲃细胞系，细胞形态与其他鱼类相似，为成纤维样细胞，只是生长周期略长，细胞鉴定的结果表明细胞系来自于滇池金线鲃，且滇池金线鲃成纤维样细胞可接受外源基因，SGVF细胞系已传至50多代，其他细胞系传至20多代，均已液氮保存。（2）滇池金线鲃的细胞培养中，引入了种群的概念，滇池金线鲃鳍条细胞系下属的几株细胞来源于不同的地理种群（牧羊河居群和旧寨龙潭居群）和同一地理种群的不同世代（牧羊河种鱼居群、子一代和子二代）。（3）将滇池金线鲃细胞培养方案改良并应用于其他云南珍稀鱼类，建立了7种鱼类的9株细胞系，包括鱇浪白鱼Anabarilius grahami、星云白鱼Anabarilius andersoni、花（鱼骨）Hemibarbus maculatus、高体鰟鮍Rhodeus ocellatus、滇池金线鲃、抚仙金线鲃Sinocyclocheilus tingi、鸭嘴金线鲃Sinocyclocheilus anatirostris、暗色唇鱼Semilabeo obscurus。 尽管滇池金线鲃人工繁殖成功，但塘养环境下，像其他养殖鱼类一样，滇池金线鲃也会出现雌雄发育不同步的现象，雄鱼成熟约早于雌鱼一个月，而精子冷冻保存技术解决这一问题，不仅可实现精子和卵子的同步化，也可保存其种质资源。鉴于此，我们开展了滇池金线鲃的精子冷冻保存研究，（1）建立了滇池金线鲃精子超低温冷冻技术（稀释液：D-15（136.75mM NaCl，6.71mM KCl，83.33 mM Glucose）；抗冻剂：7.5%乙二醇；稀释比例（精液体积：稀释液体积）：1:2；稀释方式：稀释液等体积分成两份，分别稀释精液，间隔5min；平衡时间：60min；降温速率：液氮面上6cm，2min，液氮面上2min，然后入液氮），解冻后精子活力约为40%，不到鲜精的一半，但胚胎发育各时期冻精的胚胎存活率与鲜精无显著差异。（2）此技术同样适用于滇池金线鲃子一代精子的冷冻保存，因此，精子冷冻保存过程中加入了种群概念。（3）借鉴滇池金线鲃的精子超低温冷冻保存技术，改善后并用于其它云南珍稀土著鱼类，研究了软鳍新光唇鱼Neolissochilus benasi和暗色唇鱼的精子冷冻保存技术，取得较好效果，揭示了精子超低温冷冻技术在以滇池金线鲃为代表的云南珍稀鱼类保护中的应用潜力，同时也揭示了亲鱼培育对于精子质量的重要性，进而影响精子超低温冷冻保存的成败。（4）精子冷冻保存技术作为一种精子筛选手段，能否成为遗传育种的重要手段还有待进一步研究，比如它是否影响了滇池金线鲃的幼苗生长、发育，是否降低了种群的遗传多样性等。 通过滇池金线鲃精子超低温冷冻保存的研究发现，精子质量评价体系的建立至关重要。建立了滇池金线鲃的精子质量评价体系，主要包括精子产量（精子密度、体积和总量）、活力、膜完整率、激活后运动持续时间（快速运动时间、慢速运动时间和寿命），精子形态学测量（头直径和尾长）等指标，并探索了采集日期、精液来源、雄鱼年龄和催产剂对精子质量的影响，结果显示（1）采集日期、精液来源、雄鱼年龄和催产剂均可影响精子质量，但对精子形态（头直径和尾长）的影响不大。（2）采集日期通过影响雄鱼的成熟率从而影响精子质量；精液来源对精子质量的影响则是亲鱼培育和对塘养环境的适应联合作用的结果；随着雄鱼年龄的增加，精子质量逐渐下降，而亲鱼培育的增强可减缓这一过程，使精子质量维持在较高的水平。（3）催产剂的使用提高了精液产量，而降低了精子活力和激活后运动持续时间，因此，在渔业生产中，催产剂并不是万能的，应将目光投向亲鱼培育，通过亲鱼培育的不断加强，可实现养殖鱼类的自然产卵、产精，无需借助催产剂。（4）滇池金线鲃精子质量评价体系也使用于其他云南土著珍稀鱼类，用以评价其精子质量，以指导生产实践。 |
| 英文摘要 | Sinocyclocheilus grahami (Regan, 1904), a troglophile species, is only distributed in Dianchi Lake, Yunnan, China. As a commercial fish species, it was once abundant in natural water bodies and valued for its attractive body color and tasty flesh, but its yield has critically declined because of pollution and overharvesting. S. grahami has now been categorized into the second class of special state protection on the national wildlife protection list and the China Species Red List, Vol. 1 and as critically endangered on the IUCN Red List. To protect this valuable species, we have been making some advances into the artificial propagation of S. grahami and achieved success in 2007. Artificial fertilization protect S. grahami on individual and population level, and few studies about cell level have been reported. Some continuous cell line from fin, heart and saccus olfactorius of S. grahami has been established and characterized. The SGVF cell line consists of fibroblast-like cells and has been subcultured more than 50 times over three years. The cell line was maintained in L-15 supplemented with 10% FBS at 20°C, and the double time was 73h. Cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15. The growth rate of S. grahami fin cells increased when the FBS proportion increased from 5% to 20% at 20°C with the optimal growth at the concentrations of 20% FBS. The cells could grow at temperature between 15 and 20°C with the optimal temperature of 20 °C. DMSO is the better cryoprotectant for SGVF cells with the optimal concentration of 2.5% DMSO. The distribution of chromosome number was varying from 48 to 103 with a modal peak at 96 chromosomes, accounted for 47.1% of all cells. Sequences of PCR products were identical no matter obtained from SGVF cells or muscle tissues of S. grahami. When the cells were transfected with pEYFP-N1 and pECFP-N1 plasmid, bright ?uorescent signals were observed, which suggesting that this cell line is suitable for transfection and gene expression studies. The cell culture technology of S. grahami was also suitable for other valuable freshwater fish from Yunnan province, and nine cell lines have been established from Anabarilius grahami、A. andersoni、Hemibarbus maculatus、Rhoaeus ocellatus、S. grahami tingi、S. anatirostris、Semilabeo obscurus. Similar to other cultured fish, there are intractable problems with the artificial propagation and broodstock management of S. grahami, such as decreased genetic diversity as the frequency of artificial propagation increased, asynchrony in the gamete availability of both sexes, and lower sperm quality in cultured fish than wild fish. For farmed S. grahami, males mature a month earlier than females, and sperm cryopreservation is applied to achieve the synchronization of artificial reproduction and the maintenance of the genetic variability of the original fish stocks. Thus, to further protect and conserve the genetic resource of S. grahami, sperm cryopreservation is particularly important for the continued development of S. grahami aquaculture. Following the successful artificial propagation of S. grahami, this study developed a protocol for the cryopreservation of sperm to further protect and conserve the genetic resource of this species. Sperm was collected from twenty-four mature males. A single-factor design was applied to study the effects of extender, cryoprotectant, dilution ratio, method of dilute, equilibration time and colling rate on the motility of post-thaw sperm stored at 4℃ and in liquid nitrogen. The toxicity of cryoprotectants on the motility of fresh sperm was also evaluated. Higher post-thaw sperm motility was gained with extender D-15 (83 ± 6% and 39 ± 5% for 4℃ and liquid nitrogen, respectively). Based on its toxicity and cryoprotective efficiency, 7.5% EG is a suitable cryoprotectant for S. grahami sperm. The optimal dilution ratio is 1:2, and equilibration time is 60 min. Samples were frozen by first being placed 6 cm above liq |
| 语种 | 中文 |
| 公开日期 | 2014-06-18 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7906]  |
| 专题 | 昆明动物研究所_系统进化与生物地理学 昆明动物研究所_东南亚野生动物多样性 |
| 推荐引用方式 GB/T 7714 | 王晓爱. 超低温冷冻技术在滇池金线鲃种质资源保存中的应用[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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